Photoreceptors not only receive light as an input, they also get feedback signals from horizontal cells (HCs). Negative feedback from HCs is believed to be the fundamental mechanism underlying the center-surround organization of the receptive field of retinal neurons. The mechanism responsible for HC feedback is controversial, but one major hypothesis is that the calcium current at the cone terminal is regulated by the acidity (i.e. pH) of the synaptic cleft. Although the pH hypothesis is currently very popular, it is still unknown if the pH value of the synaptic cleft really changes under physiological conditions, and if this change is significant enough to mediate the feedback. To address these questions, transgenic zebrafish expressing a pH-sensitive GFP variant either on HC or cone surfaces have been created in order to monitor the pH within the synaptic invagination. Patterned illumination will be transmitted onto the retina while membrane fluorescence is monitored. A pH change within the cone synaptic cleft will be reflected by a corresponding change in fluorescence intensity, and the direction and magnitude of the fluorescence drift will be compared to the expected pH change required for mediating feedback responses. Recently, an unexpected positive feedback mechanism from HCs to cones was discovered, which presumably boosts the gain of photoreceptors. This positive feedback is likely initiated by calcium influx into HCs, which subsequently accelerates neurotransmitter release from cones through an unknown mechanism. To examine the importance of HC calcium influx in initiating this feedback, transgenic zebrafish expressing GluR2 in HCs have been created. Because GluR2-containing glutamate receptors are calcium-impermeable, positive feedback is expected to be attenuated. To test if calcium alone is sufficient to elicit positive feedback, transgenic zebrafish expressing TRPV1 channels in HCs will be created. TRPV1 channels are calcium- permeable, therefore, if calcium itself is sufficient to initiate positive feedback, a TRPV1 agonist should trigger the positive feedback as well. This proposal addresses long standing debates in negative feedback and provides clues for understanding the newly discovered positive feedback in visual signal processing. Understanding how feedback signals are given to cone cells will help vision scientists design better interventions, such as gene therapy or prosthetic devices, to aid in restoring vision. PUBLIC HEALTH RELEVANCE: Relevance Understanding how visual signals are processed in the retina will help vision scientists to design better interventions like gene therapy or prosthetic devices for restoring vision.